In the method for solid culture of microorganisms and the like, solid media using agar or agarose as a solidifying agent have been conventionally used. This is because there are advantages such as those that production is easy, cultured microorganisms can be easily discriminated by visual inspection, and isolation of microorganisms can be easily carried out. However, these solid media are such that the gel is dissolved in a severe environment of high temperature, extreme pH, and the like, therefore the microorganisms that can be cultured are limited.
In order to enable culture of microorganisms in such an extreme environment, a cellulose gel medium which employs cellulose only as a solidifying agent has been developed. For example, Patent Document 1 (WO 2005/083056) describes a solid medium containing, as a medium solidifying component, a cellulose gel which is a porous cellulose gel structure having a backbone moiety composed of cellulose and having a cellulose concentration of 0.01% or greater and a porosity of 50% or greater, and a method for the production thereof. This solid medium of the invention is produced by dispersing cellulose in a solvent (particularly, an aqueous thiocyanate solution), dissolving cellulose therein under stirring and/or heating, subsequently gelling the solution by cooling and/or solvent removal to obtain a cellulose gel, and causing nutrients to penetrate into the cellulose gel. Since this cellulose gel is produced by using unmodified cellulose as a raw material, the cellulose gel exhibits strong resistance even to heat, extreme pH, high salt concentrations, solubility in water or organic solvents, and the like. Accordingly, the cellulose gel medium thus produced is such that the gel does not dissolve even in an environment called an “extreme environment” in the field of microbial culture. Therefore, it is described that this medium can be used under a wider range of culture conditions in which a conventional solid medium such as an agar medium cannot be used.
As such, solid media that use cellulose gel have an excellent advantage that culture can be carried out without softening or syneresis even under the conditions in which culture is difficult in a solid medium using agarose gel, gellan gum or silica gel, for example, at a high temperature of 100° C. or under strongly acidic or strongly alkaline conditions. However, it is described in Non-Patent Document 1 (Deguchi et al., Soft Matter, (2007), 3, 1170-1175) that in order to produce a cellulose gel medium which is appropriate for microbial culture, the optimal cellulose concentration is 2% to 3% by weight, and if the concentration is less than 1% by weight, the gel loses its physical strength and becomes very brittle, so that it is difficult to apply or disperse microorganisms thereon. Furthermore, it is described that if the cellulose concentration is greater than 3% by weight, the medium surface after solidification does not become flat, and inoculation of microorganisms cannot be achieved. Therefore, production of cellulose gel media for microbial culture is highly limited under the current circumstances.
Furthermore, a cellulose gel having a cellulose concentration which is suitable as a solid medium in view of physical strength or smoothness, such as described above, does not have such transparency as that of agar or agarose gel media and is white in color. Since most of the microbial colonies are whitish, visibility of the colonies on cellulose gel media is poor, and cellulose gel media are not suitable for the observation of colonies. However, there has been no alternative to improve this situation.
Cellulose gel media are also useful for the screening of cellulase-producing bacteria. Conventionally, the screening of cellulase-producing bacteria has been carried out by a method of using carboxymethyl cellulose (CMC) or the like, but as a result of the use of cellulose gel media, the screening efficiency has dramatically improved as compared with conventional methods. This improvement is based on the reason that since cellulose, which is the material of cellulose gel media, is a direct substrate of cellulases, when cellulose is decomposed, the presence of cellulase-producing bacteria at the place of decomposition can be visually determined, and therefore, a separate operation for confirming the cellulase activity is not required, and that production of replicas for screening is not required.
In regard to screening, since an increase in the efficiency of operation, particularly shortening of the detection time, greatly affects the development status thereafter, a speedup of screening has been constantly desirable. Decomposition of cellulose gel by a cellulase is such that the influence of the decomposition can be more clearly observed as the cellulose density is lower. However, in the current situation, since it has been difficult to reduce the cellulose concentration in the cellulose gel, further speedup could not be desired. Furthermore, since cellulose media are white in color, checking of holes produced by decomposition has been difficult in the early stage of culture.